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Results for “mutational profiling”

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PubMed2002

Switch recombination in fetal porcine thymus is uncoupled from somatic mutation.

Since fetal serum Ig isotype profiles suggested that IgG and IgA could be of de novo origin, we studied their transcription and secretion. IgM transcripts were present at 50 days of gestation in major fetal lymphoid tissues, IgG and IgA transcription was pronounced at 60 days in fetal thymus and both transcription and secretion in this organ increased in late fetal life. The CDR3 spectratype of thymic IgG and IgA transcripts was as polyclonal as that of IgM already at 70 days in utero indicating a broad repertoire of switched B-cells. However, VDJs transcribed with the switched isotypes were not hypermutated as were those from immunized fetuses, indicating that switch recombination and somatic mutation are not coupled in utero in piglets. This finding and the fact that the oligoclonal IgA and IgM repertoires in a non-inductive site of the mucosal immune system (parotid gland) becomes polyclonal in piglets reared germ-free, suggest that initial expansion of switched B-cells in fetal and neonatal piglets is not driven by environmental antigen. Our findings collectively suggest that all IgA and IgM may result from de novo synthesis while some IgG probably results from selective transport. The latter is consistent with the gradual decline in serum IgG concentration in germ-free isolator piglets and the expression of FcRn in the porcine placenta.

Animals↗

PubMed1993

Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes.

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.

Base Sequence↗

PubMed2004

Effect of mutated transporters associated with antigen-processing 2 on characteristic major histocompatibility complex binding peptides: analysis using electrospray ionization tandem mass spectrometry.

A novel allele of transporters associated with the antigen-processing (TAP) 2 gene, TAP2*Bky2 (Val(577)), is significantly increased in Japanese patients with Sjögren's syndrome (SS), and has a strong association with SS-A/Ro autoantibody production in SS and autoantibody including anti-SS-A/Ro and anti-U1 RNP antibody in systemic lupus erythematosus (SLE). To determine the influence of this natural mutated TAP on peptides loaded onto MHC class I, we analyzed the repertoire of peptides loaded onto MHC class I on transfectants with TAP1 and TAP2 or mutated TAP2 by electrospray ionization tandem mass spectrometry (ESI-MS/MS). After comparison of the peptide profiles we identified three peptides from only mutated TAP transfectants. Moreover, one of these peptides is derived from snRNP A, which is a target for anti-U1 RNP antibody. To our knowledge this is the first report to show that the natural mutation of TAP2 changes the peptide profile loaded onto MHC class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2025

Small cell bladder carcinoma with a high tumor mutational burden responding to sequential cisplatin-etoposide and pembrolizumab: a case report.

Small cell carcinoma of the urinary bladder (SCCB) is a rare and aggressive malignancy with limited treatment options and a poor prognosis. We present the case of a 63-year-old man who was initially diagnosed to have non-metastatic high-grade non-muscle invasive urothelial carcinoma with sarcomatoid subtype and later developed bone metastases. A bone biopsy confirmed small cell carcinoma, and retrospective review of the original tumor revealed mixed histology comprising small cell, sarcomatoid-like, and conventional urothelial carcinoma components. The patient was treated with six cycles of cisplatin and etoposide, during which genomic profiling identified a high tumor mutational burden (22 mutations/megabase). Based on this finding, pembrolizumab was administered sequentially as monotherapy. The patient achieved a complete response that lasted for more than 1 year, but subsequently developed lymph node metastases and recurrences in bone. This case highlights the role of genomic profiling test for clinical decision-making as tumor mutational burden predicts the efficacy of immune checkpoint inhibitor therapy in SCCB. This case also underscores the urgent need for novel treatment approaches for SCCB.

Case report↗

PubMed2026

Post-Hoc Long-Read Sequencing Links Leukemic Mutation Status to Single-Cell Transcriptomes.

Single-cell RNA-sequencing-based characterization of cells that belong to the neoplastic clone is a major challenge in hematologic neoplasms, where malignant and normal cells coexist. Confident molecular profiling requires simultaneous analysis of gene expression and genetic mutations in individual cells, an ability that is not supported by the standard 10X Genomics workflow. Here, we systematically evaluated the potential and limitations of repurposing amplified cDNA generated during the 10X Genomics 3' workflow for post hoc genotyping of individual cells. We first established a mixed leukemic cell line system comprising one cell line with KIT point mutations and another with the BCR::ABL1 fusion gene. Targeted long-read PacBio sequencing enabled post hoc assignment of mutation data to transcriptionally profiled cells, but recovery differed between targets. Consistent with ambient RNA in microfluidics-based single-cell workflows, mutation-associated transcripts were detected in cells not expected to carry the corresponding mutations, illustrating how transcript recovery complicates cell-level genotype assignment. Target-specific thresholds mitigated this source of misclassification. In primary chronic myeloid leukemia samples, the post hoc approach detected BCR::ABL1-positive cells at diagnosis, but not during imatinib treatment. Together, we present a framework for adding mutation status to cells already profiled using the 10X Genomics workflow and highlight broader considerations for transcript-based single-cell genotyping.

BCR::ABL1↗

PubMed2006

Patterns of hepatitis B virus infection in Brazilian human immunodeficiency virus infected patients: high prevalence of occult infection and low frequency of lamivudine resistant mutations.

Hepatitis B virus (HBV) molecular profiles were determined for 44 patients who were infected with human immunodeficiency virus (HIV) type 1 and had antibodies to the hepatitis B core antigen (anti-HBc), with and without other HBV serological markers. In this population, 70% of the patients were under lamivudine treatment as a component of antiretroviral therapy. HBV DNA was detected in 14 (32%) patients. Eight out of 12 (67%) HBsAg positive samples, 3/10 (30%) anti-HBc only samples, and 3/22 (14%) anti-HBs positive samples were HBV DNA positive. HBV DNA loads, measured by real time polymerase chain reaction, were much higher in the HBsAg positive patients (mean, 2.5 x 10(9) copies/ml) than in the negative ones (HBV occult infection; mean, 2.7 x 10(5) copies/ml). Nine out of the 14 HBV DNA positive patients were under lamivudine treatment. Lamivudine resistant mutations in the polymerase gene were detected in only three patients, all of them belonging to the subgroup of five HBsAg positive, HBV DNA positive patients. A low mean HBV load (2.7 x 10(5) copies/ml) and an absence of lamivudine resistant mutations were observed among the cases of HBV occult infection.

Adult↗

PubMed2006

Molecular profiling of bladder tumors based on the detection of FGFR3 and TP53 mutations.

PURPOSE: On a routine basis we performed systematic molecular screening for FGFR3 and TP53 mutations in 121 bladder tumors. We then specifically analyzed the predictive value of the recurrence of FGFR3 and TP53 genotypes in superficial lesions. MATERIALS AND METHODS: The FGFR3 gene was analyzed by direct sequencing of exons 7, 10 and 15, whereas TP53 status was determined using the p53 functional assay in yeast. RESULTS: We identified a missense FGFR3 mutation in 66% of pTa, 26% of pT1 and 12% of pT2 tumors. Of activating FGFR3 mutations 54% and 85% were found in low G1 and intermediate G2 grade tumors, respectively, but in only 20% of high grade G3 tumors. We detected inactivating TP53 mutations in 10% of pTa, 42% of pT1 and 58% of pT2 tumors. Moreover, TP53 mutations were found only in 23% of grade G1 and 3% of grade G2 tumors but in 44% of high grade G3 tumors. When the 2 genotypes were combined, we observed that 58% of pTa tumors had the (mutant FGFR3, WT TP53) genotype, whereas 58% of invasive lesions harbored the inverse genotype (WT FGFR3, mutant TP53). The (mutant FGFR3, WT TP53) genotype and the (WT FGFR3, mutant TP53) genotype were detected in 23% and 38% of pT1G3 tumors, respectively. In the subgroup of 92 patients with superficial pTa-T1 bladder tumors we did not find that the TP53 or FGFR3 genotype alone or combined had a predictive value for tumor recurrence. CONCLUSIONS: Our data again represent solid proof for the pivotal role of FGFR3 and TP53 mutations in superficial and invasive bladder tumors, respectively. However, other molecular markers should be identified for borderline pT1G3 bladder tumors, which are probably at the crossroads of these 2 distinct molecular pathways.

Aged↗

PubMed1991

Inhalation exposure in Drosophila mutagenesis assays: experiments with aliphatic halogenated hydrocarbons, with emphasis on the genetic activity profile of 1,2-dichloroethane.

A series of mutation experiments was carried out with Drosophila melanogaster using inhalation exposure. 1,2-Dichloroethane (DCE) and 1,2-dibromoethane (DBE) were active in the sex-linked recessive lethal assay (SLRLT), whereas dichloromethane, dibromomethane, 1,2-dichloropropane and 1,3-dichloropropane were not. Compared to DBE, DCE is a less potent mutagen in the SLRL system. For both compounds, there is no evidence of a clear-cut dose-rate effect. DCE and dichloromethane were also investigated in the somatic mutation and recombination test (SMART), with results similar to those from the SLRLT. For DCE the genetic activity profile was further analyzed by carrying out a sex-chromosome loss assay and a complementation analysis of a series of induced recessive lethal mutations. A review of the use of inhalation in mutagenicity assays with Drosophila shows that this route of exposure is an effective one. Especially with chronic exposure times, rather low exposure concentrations can be detected. With compounds of intermediate volatility inhalation is not superior to other modes of administration; nor is it likely to be sensitive enough for in situ monitoring.

Administration, Inhalation↗

PubMed1994

Characterization of rifampin-resistance in pathogenic mycobacteria.

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.

Amino Acid Sequence↗

PubMed2005

Comprehensive characterization of HNPCC-related colorectal cancers reveals striking molecular features in families with no germline mismatch repair gene mutations.

A considerable fraction of families with HNPCC shows no germline mismatch repair (MMR) gene mutations. We previously detected 'hidden' MMR gene defects in 42% of such families, leaving the remaining 58% 'truly' mutation negative. Here, we characterized 50 colorectal carcinomas and five adenomas arising in HNPCC families; 24 truly MMR gene mutation negative and 31 MMR gene mutation positive. Among 31 tumors from MMR gene mutation positive families, 25 (81%) had active Wnt signaling as indicated by aberrant beta-catenin localization with or without CTNNB1 mutations, compared to only 7/18 tumors from MMR gene mutation negative families (39%; P=0.005). CGH studies revealed stable profiles in 9/16 (56%) of MMR gene mutation negative tumors, which was significantly associated with membranous beta-catenin (P=0.005). Tumors with membranous beta-catenin from the MMR gene mutation negative group also showed low frequency of TP53 mutations compared to those with nuclear beta-catenin. Thus, a majority of the MMR gene mutation negative cases exhibited a novel molecular pattern characterized by the paucity of changes in common pathways to colorectal carcinogenesis. This feature distinguishes the MMR gene mutation negative families from both HNPCC families linked to MMR defects and sporadic cases, suggesting the involvement of novel predisposition genes and pathways in such families.

Base Pair Mismatch↗

PubMed2002

The pathology of familial breast cancer: predictive value of immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in patients with mutations in BRCA1 and BRCA2.

PURPOSE: The morphologic and molecular phenotype of breast cancers may help identify patients who are likely to carry germline mutations in BRCA1 and BRCA2. This study evaluates the immunohistochemical profiles of tumors arising in patients with mutations in these genes. MATERIALS AND METHODS: Samples of breast cancers obtained from the International Breast Cancer Linkage Consortium were characterized morphologically and immunohistochemically using antibodies to estrogen receptor, progesterone receptor, HER-2 (c-erbB-2 oncogene), and p53 protein. RESULTS: Breast cancers in patients with BRCA1 germline mutations are more often negative for estrogen receptor, progesterone receptor, and HER-2, and are more likely to be positive for p53 protein compared with controls. In contrast, BRCA2 tumors do not show a significant difference in the expression of any of these proteins compared with controls. CONCLUSION: BRCA1 has a distinctive morphology and immunohistochemical phenotype. The combined morphologic and immunohistochemical data can be used to predict the risk of a young patient harboring a germline mutation in BRCA1. The BRCA2 phenotype is currently not well defined.

Adult↗

PubMed2006

Target genes of the WNT/beta-catenin pathway in Wilms tumors.

The WNT/beta-catenin pathway is involved in numerous human cancers. Mutations of the CTNNB1 (beta-catenin) gene have also been detected in a subset of pediatric Wilms tumors, but the target genes of the deregulated WNT/beta-catenin pathway in these tumors have yet to be identified. To compare gene expression profiles of Wilms tumors with and without mutations of CTNNB1, we used 11.5-k cDNA microarrays. Most of the tumors (86%) had received preoperative chemotherapy as mandated by the European SIOP protocol. The comparison between Wilms tumors with and without CTNNB1 mutations revealed several target genes specifically deregulated in CTNNB1-mutated Wilms tumors. Among these, PITX2, APCDD1, and two members of the endothelin axis (EDN3 and EDNRA) are directly activated downstream targets of the WNT/beta-catenin pathway that may enhance proliferation of these tumor cells. In addition, several upstream inhibitors of WNT/beta-catenin signaling like WIF1 and PRDC were also strongly up-regulated in the CTNNB1-mutated Wilms tumors. This overexpression may be a negative feedback mechanism in tumors with uncontrolled WNT signaling. Moreover, we identified deregulated genes in both the retinoic acid and the RAS pathways, such as ATX/ENPP2 and RIS1, suggesting an association between these two pathways with that of WNT. In addition, the strong representation of muscle-related genes in the expression profile of CTNNB1-mutated Wilms tumors corresponded to histologically detectable areas of myomatous cells in these tumors that displayed intense and preferential nuclear beta-catenin antibody staining. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

DNA, Complementary↗

PubMed2002

Genetic progression in sporadic endometrial and gastrointestinal cancers with high microsatellite instability.

This study selected a series of 136 MSI-H (microsatellite instability at high frequency) gastric, colorectal, and endometrial carcinomas combining immunohistochemical analysis for hMLH1 or hMSH2 gene products and microsatellite study. The clinico-pathological profile of all tumours was correlated with the overall instability rates at coding and non-coding repeats, in order to clarify the role and the mutation timing of seven target genes (TGFbetaRII, IGFIIR, BAX, hMSH3, hMSH6, CHK1, and BRCA2) in the progression of an MSI-H neoplasm. Regardless of the primary site, the results confirm a model of oncogenesis in which inactivation of hMLH1, or less frequently hMSH2, may initiate a pathway culminating in a progressive accumulation of frameshifts in coding region (CDR) microsatellites. Comparing gastrointestinal and endometrial tumours, significantly lower levels of microsatellite instability at both coding and non-coding repeats were observed. Among gastric and colorectal tumours, the detection of small shortening within Bat-26 and Bat-25 markers defines a subgroup of MSI-H gastrointestinal tumours invariably characterized by early stage at diagnosis. In these tumours, mutations of TGFbetaRII or BAX genes precede frameshifts in the other tested genes. The analysis of correlations between the mutational and clinico-pathological profiles of advanced gastrointestinal tumours revealed that the higher levels of microsatellite instability at both coding and non-coding repeats were not associated with a more advanced clinico-pathological stage or a less favourable outcome. A significant association was observed between a low number of CDR frameshifts and the presence of lymph-node metastasis in advanced gastrointestinal tumours. The existence of advanced MSI-H tumours with more aggressive behaviour and a 'mild mutator phenotype' could be explained by hypothesizing an overlapping of different mechanisms of tumourigenesis, including both the mutator and the suppressor pathways; this should be tested by further studies.

Cell Transformation, Neoplastic↗

PubMed2000

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Cell Wall↗

PubMed2001

A novel mutation (N233K) in the transactivating domain and the N756S mutation in the ligand binding domain of the androgen receptor gene are associated with male infertility.

OBJECTIVE: Resistance to androgens has been suggested as a possible cause of male infertility. This hypothesis is based mainly on binding studies in genital skin fibroblasts but the molecular evidence is sparse. DESIGN: Molecular studies of the androgen receptor gene were performed in 10 azoo- or oligozoospermic men, presenting with clinical signs of low androgen activity-poor virilization and high serum LH despite elevated testosterone levels, but without genital malformations. PATIENTS: Ten men with serum LH >10 IU/l and testosterone >30 nmol/l as well as a low sperm concentration < 20 x 106/ml. MEASUREMENTS: Genomic DNA was prepared from peripheral leucocytes and PCR-amplification of the coding region of androgen receptor was performed, followed by direct sequencing. Identified mutations were reconstructed by site-directed mutagenesis and the functional properties of the mutants were analysed, using transient expression in COS-1 cells and subsequent transactivation assays. Hormone binding assays were performed in genital skin fibroblasts from the patients. RESULTS: Two of the 10 men were shown to have a mutation in the androgen receptor gene. Subject 1, who presented with azoospermia, serum testosterone (T) 50 nmol/l and LH 20 IU/l, had a mutation in exon 1, changing amino acid asparagine 233 to lysine (N233K). In fibroblasts cultured from genital skin, the receptor affinity for 5alpha-dihydrotestosterone (DHT) was normal as compared to healthy controls, but the receptor-hormone complex was thermolabile at 42 degrees C. Subject 2 exhibited severe oligozoospermia and a similar endocrine pattern (T = 50 nmol/l and LH = 25 IU/l). He had a mutation in exon 5 changing asparagine 756 to serine (N756S). The affinity for DHT in cultured genital fibroblasts from this patient was reduced. Transactivation was abnormal for both mutants, N233K reaching 46% and N756S 38% of wild type activity when stimulated with 10 nmol/l DHT. CONCLUSIONS: Androgen receptor mutations may affect sperm production without resulting in genital malformations. Thus, in infertile men with a clinical presentation of poor androgen activity and an endocrine profile compatible with androgen resistance, mutations in the androgen receptor should be taken into consideration.

Animals↗

PubMed2006

[Analysis of ethambutol drug-resistance gene mutation in Mycobacterium tuberculosis by specific endonuclease].

OBJECTIVE: Surveyor nuclease is a member of the family of plant endonucleases that cleave heteroduplexes DNA with high specificity at sites of base substitution mismatch and DNA distortion. Heteroduplex analysis by Surveyor nuclease is a relatively new method. The purpose of this study was to explore the application of this method in analysis of drug-resistance gene mutation in Mycobacterium tuberculosis. METHODS: Surveyor nuclease was used to analyze embB gene mutation of 60 Mycobacterium tuberculosis isolates, among which 45 were EMB-resistant and embB gene mutation was found by sequencing, and 15 EMB-susceptible isolates without embB mutation. PCR amplification was carried out first, and then hybridized with products of H37Rv to form heteroduplex, cleaved by Surveyor nuclease, and lastly the results were shown by polyacrylamide gel electrophoresis and mutation was judged according to electrophoresis profile. RESULTS: Forty-five EMB-resistant isolates were found harboring embB gene mutation, while in the 15 EMB-susceptible isolates no mutation was found. All the 45 EMB-resistant isolates were revealed to harbor embB gene hotspot codon 306 mutation, among which 33 with ATG-->GTG, 3 with ATG-->ATT, 5 with ATG-->ATA, 2 with ATG-->ATC, 2 with ATG-->CTG. These results from Surveyor nuclease method was completely parallel to those of sequencing. CONCLUSION: Heteroduplex analysis by Surveyor nuclease may become a useful method for drug-resistance gene mutation analysis for Mycobacterium tuberculosis because of its simplicity, stability, low-cost, and high sensitivity and specificity.

Antitubercular Agents↗

PubMed1994

Insulin secretory abnormalities in subjects with hyperglycemia due to glucokinase mutations.

Pancreatic beta-cell function was studied in six subjects with mutations in the enzyme glucokinase (GCK) who were found to have elevated fasting and postprandial glucose levels in comparison to six normoglycemic controls. Insulin secretion rates (ISRs) were estimated by deconvolution of peripheral C-peptide values using a two-compartment model and individual C-peptide kinetics obtained after bolus intravenous injections of biosynthetic human C-peptide. First-phase insulin secretory responses to intravenous glucose and insulin secretion rates over a 24-h period on a weight maintenance diet were not different in subjects with GCK mutations and controls. However, the dose-response curve relating glucose and ISR obtained during graded intravenous glucose infusions was shifted to the right in the subjects with GCK mutations and average ISRs over a glucose range between 5 and 9 mM were 61% lower than those in controls. In the controls, the beta cell was most sensitive to an increase in glucose at concentrations between 5.5 and 6.0 mM, whereas in the patients with GCK mutations the point of maximal responsiveness was increased to between 6.5 and 7.5 mM. Even mutations that resulted in mild impairment of in vitro enzyme activity were associated with a > 50% reduction in ISR. The responsiveness of the beta cell to glucose was increased by 45% in the subjects with mutations after a 42-h intravenous glucose infusion at a rate of 4-6 mg/kg per min. During oscillatory glucose infusion with a period of 144 min, profiles from the subjects with mutations revealed reduced spectral power at 144 min for glucose and ISR compared with controls, indicating decreased ability to entrain the beta cell with exogenous glucose. In conclusion, subjects with mutations in GCK demonstrate decreased responsiveness of the beta cell to glucose manifest by a shift in the glucose ISR dose-response curve to the right and reduced ability to entrain the ultradian oscillations of insulin secretion with exogenous glucose. These results support a key role for the enzyme GCK in determining the in vivo glucose/ISR dose-response relationships and define the alterations in beta-cell responsiveness that occur in subjects with GCK mutations.

Adolescent↗

PubMed2006

Genetic pleiotropy in Saccharomyces cerevisiae quantified by high-resolution phenotypic profiling.

Genetic pleiotropy, the ability of a mutation in a single gene to give rise to multiple phenotypic outcomes, constitutes an important but incompletely understood biological phenomenon. We used a high-resolution and high-precision phenotypic profiling approach to quantify the fitness contribution of genes on the five smallest yeast chromosomes during different forms of environmental stress, selected to probe a wide diversity of physiological features. We found that the extent of pleiotropy is much higher than previously claimed; 17% of the yeast genes were pleiotropic whereof one-fifth were hyper-pleiotropic. Pleiotropic genes preferentially participate in functions related to determination of protein fate, cell growth and morphogenesis, signal transduction and transcription. Contrary to what has earlier been proposed we did not find experimental evidence for slower evolutionary rate of pleiotropic genes/proteins. We also refute the existence of phenotypic islands along chromosomes but report on a remarkable loss both of pleiotropy and of phenotypic penetrance towards chromosomal ends. Thus, the here reported features of pleiotropy both have implications on our understanding of evolutionary processes as well as the mechanisms underlying disease.

Genes, Fungal↗